Influenza virus infection in humans is a respiratory disease that ranges in severity from subclinical infection to primary viral pneumonia that can result in death. Influenza-associated complications include, among others, Reye's syndrome, myocarditis, pericarditis, myositis, encephalopathy and transverse myelitis. The persistence and unfettered nature of influenza virus leads to yearly epidemics as well as sporadic pandemics with potential to cause catastrophic loss of life. Palese et al., Nature Medicine 8(9):927 (2002). Seasonal influenza is the seventh leading cause of death in the United States and the leading cause of death in children ages 1 to 4 years. Ninety percent of deaths in people 65 and older are the result of influenza virus infection with associated pneumonia. Every year in the United States, approximately 36,000 people die, 114,000 are hospitalized, and the country incurs more than $1 billion in direct economic costs.
Three types of influenza viruses (A, B, and C) are distinguishable by antigenic reactivities of their internal antigens. Influenza A, B and C belong to the family Orthomyxoviridae and have a segmented negative strand RNA genome that is replicated in the nucleus of the infected cell and consists of eight negative-sense RNA (nsRNA) gene segments that encode 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA, which after enzymatic cleavage is made up of the association of subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2, also referred to as Nuclear Export Protein (NEP)). Krug et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York, 1989, pp. 89-152. The HA and NA proteins embedded in the viral envelope are the primary antigenic determinants of the influenza virus (Air et al., Structure, Function, and Genetics, 1989, 6:341-356; Wharton et al., In The Influenza Viruses, R. M. Krug, ed., Plenum Press, New York, 1989, pp. 153-174). Due to the possible reassortment of the influenza virus' segmented genome (antigenic shift) and the accumulation of genomic polymorphisms (antigenic drift), new HA and NA variants are constantly created for which a newly infected organism has no anamnestic immune response. Such constant generation of new antigenic variants from a vast number of circulating strains creates enhanced danger of emergence of new highly pathogenic strains (such as, e.g., H5N1 and H1N1 influenza A virus transmitted directly from avian or swine species to humans) and creates the need for annual vaccination and development of antiviral agents that are effective against many or all strains. Palese, Nature Medicine 10(12 Suppl):S82 (2004); Garcia-Sastre and Biron, Science 312(5775):879 (2006); Li et al., Nature 2004, 430:209; Kuiken et al., Science 2004, 306:241. This has forced the World Health Organization to monitor current strains and constantly update the composition of the annual vaccine. For the production of a safe and effective vaccine it is important that the selected vaccine strains are closely related to the circulating strains, thereby ensuring that the antibodies in the vaccinated population are able to neutralize the antigenetically similar virus.
Among the three types of influenza viruses, influenza A and B viruses cause significant morbidity and mortality in humans. Fields et al., Lippincott Williams & Wilkins, Philadelphia, Pa., 2007. Thus, annual vaccines used to combat influenza virus infection include a combination of two influenza A strains with a single influenza B strain. Palese, Nature Medicine 10(12 Suppl):582 (2004).
Propagation of these viral strains is usually performed in embryonated chicken eggs, where the virus can grow to very high titers. The virus particles generated in eggs are subsequently purified and used as stocks for vaccine preparations. Recently, mammalian cell culture systems for large-scale influenza vaccine production have been also established. Reviewed in, e.g., Genzel and Reichl, Expert Review of Vaccines, 2009, 8(12):1681-1692. Currently, vaccines produced in three different mammalian cell lines (Madin-Darby Canine Kidney [MDCK], Vero and PER.C6) are in clinical trials.
Recently developed reverse-genetics systems have allowed the manipulation of the influenza viral genome (Palese et al., Proc. Natl. Acad. Sci. USA 1996, 93:11354; Neumann and Kawaoka, Adv. Virus Res. 1999, 53:265; Neumann et al., Proc. Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999, 73:9679; U.S. Patent Publication No. 20040029251). For example, it has been demonstrated that the plasmid-driven expression of eight influenza vRNAs from a pol I promoter and all mRNAs from a pol II promoter result in the formation of infectious influenza A virus (Hoffmann et al., Proc. Natl. Acad. Sci. USA 2000, 97:6108; Hoffmann et al., Vaccine 2002, 20:3165; U.S. Pat. No. 6,951,754).
The influenza vaccines currently licensed by public health authorities for use in the United States and Europe are inactivated influenza vaccines as well as the live attenuated FLUMIST vaccine in the United States.
Inactivated vaccines are produced by chemical inactivation of the virus grown either in cell culture or in embryonated chicken eggs. Chemical inactivation is usually followed by detergent-mediated fragmentation. Typical inactivation/fragmentation treatments involve such agents as formalin+Triton, formaldehyde, beta-propiolactone, ether, ether+Tween-80, cetyl trimethyl ammonium bromide (CTAB)+Triton N101, sodium deoxycholate and tri(n-butyl) phosphate. Nicholson, Webster and May (eds.), Textbook of Influenza, Chapters 23, 24, 27, pp. 317-332 and 358-372. For the virus produced in eggs, inactivation can occur after or prior to clarification of allantoic fluid. Although inactivation dramatically increases the safety of the vaccine, it reduces vaccine potency. Also, vaccine testing to ensure loss of replicative activity is time-consuming and labor-intensive, which increases vaccine cost and decreases the usefulness of the vaccine during rapidly spreading seasonal infections and pandemics.
Current vaccine strategies focus on live attenuated influenza virus (LAIV) strains through the development of temperature-sensitive mutants or the removal of pathogenic factors such as the NS1 protein. Talon, J. et al., Proc. Natl. Acad. Sci. USA, 97:4309-4314 (2000); Nichol, Vaccine, 19:4373-4377 (2001); Palese et al., J. Infect. Dis., 1997, 176 Suppl 1:S45-9. For example, FLUMIST (Influenza Virus Vaccine Live, Intranasal) contains influenza virus strains which are (a) cold-adapted (i.e., they replicate efficiently at 25° C., a temperature that is restrictive for replication of many wild-type influenza viruses); (b) temperature-sensitive (i.e., they are restricted in replication at 37° C. (Type B strains) or 39° C. (Type A strains), temperatures at which many wild-type influenza viruses grow efficiently); and (c) attenuated (they do not produce classic influenza-like illness in the ferret model of human influenza infection).
As compared to traditional inactivated vaccines, LAIV vaccines are well suited for mucosal (e.g., intranasal) administration and generate a more robust immune response by inducing local, mucosal, cell-mediated and humoral immunity. Treanor et al., New England J. Med. 354(13):1343 (2006) Still, current LAIV vaccines are too attenuated to stimulate a strong immune response in elderly people, the major group of the 20,000-40,000 individuals in the US dying each year as a result of influenza infection. Most importantly, present LAIV vaccines are subject to replicative impairment in embryonated chicken eggs because they have been adapted to growth at suboptimal temperatures required for proper egg development, thereby limiting the subsequent scale of vaccine production. Such impediment on global scale production must be overcome should a highly pathogenic pandemic strain emerge. Li et al., Nature 430(6996):209 (2004) and Krug, Science 311(5767):1562 (2006).
Thus, there is a great need in the art for new influenza vaccines that are safe, efficient for generating protective immunity and are amenable to rapid large-scale production in chicken eggs and/or cell culture. In particular, there is a great need in the art for new more efficient LAIV vaccines.